Enzymatic N-Allylation of Primary and Secondary Amines Using Renewable Cinnamic Acids Enabled by Bacterial Reductive Aminases

Allylic amines are a versatile class of synthetic precursors of many valuable nitrogen-containing organic compounds, including pharmaceuticals. Enzymatic allylic amination methods provide a sustainable route to these compounds but are often restricted to allylic primary amines. We report a biocatalytic system for the reductive N-allylation of primary and secondary amines, using biomass-derivable cinnamic acids. The two-step one-pot system comprises an initial carboxylate reduction step catalyzed by a carboxylic acid reductase to generate the corresponding α,β-unsaturated aldehyde in situ. This is followed by reductive amination of the aldehyde catalyzed by a bacterial reductive aminase pIR23 or BacRedAm to yield the corresponding allylic amine. We exploited pIR23, a prototype bacterial reductive aminase, self-sufficient in catalyzing formal reductive amination of α,β-unsaturated aldehydes with various amines, generating a broad range of secondary and tertiary amines accessed in up to 94% conversion under mild reaction conditions. Analysis of products isolated from preparative reactions demonstrated that only selective hydrogenation of the C=N bond had occurred, preserving the adjacent alkene moiety. This process represents an environmentally benign and sustainable approach for the synthesis of secondary and tertiary allylic amine frameworks, using renewable allylating reagents and avoiding harsh reaction conditions. The selectivity of the system ensures that bis-allylation of the alkylamines and (over)reduction of the alkene moiety are avoided.


Production and purification of SrCAR
Plasmids of carboxylic acid reductases SrCAR and plasmid of phosphopantetheine transferase (Sfp) from Bacillus subtilis were sourced from in-house plasmid collections and cloned as previously reported. [1,2] E. coli cells were transformed with CAR and Sfp plasmids.
A single colony of recombinant E. coli BL21 (DE3) containing pET28-b-FL-SrCAR and pCDF1b-Sfp was inoculated into 10 mL lysogeny broth (LB) (1% tryptone, 0.5% yeast extract, 1% NaCl) and incubated overnight at 37 °C in an orbital shaker with 200 rpm shaking. This starter culture was used as inoculum. A 2 L flask containing 500 mL LB, supplemented with kanamycin (30 μg mL -1 ) and streptomycin (50 μg mL -1 ) was inoculated with 5 mL of the starter culture. Cultivation was performed at 37 °C in an orbital shaker with 200 rpm shaking. At an optical density (OD 600 nm ) of between 0.6 and 0.8, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM to induce protein expression. Incubation was continued at 20 °C and 200 rpm for 18 h. Cells from a 500 mL culture were harvested by centrifugation at 6000 rpm for 15 min, and washed in sodium phosphate buffer (NaPi, 50 mM, pH 7.5). To isolate SrCAR, cells were disrupted by ultrasonication at 4 °C. The enzyme was isolated from the clarified lysate by Ni-affinity chromatography using a 5 mL His-Trap HP column (GE healthcare). Desalting and buffer exchange was achieved using a PD-10 desalting column (GE Healthcare, Uppsala, Sweden), eluted protein (in 50 mM NAPi, pH 7.50) was concentrated using 30 kDa cut-off Centricon filters (15 mL). Protein concentration was determined using Bradford reagents with BSA of known concentration as a reference standard. The CHU kinase [3] plasmid was sourced in-house (Turner's group) and was produced and purified using the procedure described above.

Production and purification of pIR23 and BacRedAm
pIR23 cloned into pET28a+ were sourced from Turner's group enzyme collection. [4] BacRedAm gene was codon optimised for expression in E. coli. The condon-optimised gene sequence was synthesized by Twist Bioscience (South San Francisco, CA 94080, United States) and cloned into pET28a+. E. coli cells were transformed with pET28-RedAm. A single colony of the recombinant E. coli BL21 (DE3) was inoculated into 10 mL LB and incubated overnight at 37 °C in an orbital shaker with 200 rpm shaking. This starter culture was used as inoculum for a 2 L flask containing 500 mL LB, supplemented with kanamycin (30 μg mL -1 ) was inoculated with 5 mL of the starter culture. Cultivation was performed at 37 °C in an orbital shaker with 200 rpm shaking. At an optical density (OD 600 nm ) of between 0.6 and 0.8, protein production was started with addition of IPTG to the final concentration of 0.2 mM. Incubation was continued at 20°C and 200 rpm for 18 h. Cells from a 500 mL culture were harvested by centrifugation at 6000 rpm for 15 min, and washed in sodium phosphate buffer (NaPi, 50 mM, pH 7.5). Cells were disrupted by ultrasonication at 4 °C. The RedAm was purified from the clarified lysate by Ni-affinity chromatography using a 5 mL His-Trap HP column (GE healthcare). Desalting and buffer exchange was achieved using a PD-10 desalting column (GE Healthcare, Uppsala, Sweden), eluted protein (in 50 mM NAPi buffer, pH 7.5) was concentrated using 30 kDa cut-off Centricon filters (15 mL). Concentration of isolated protein was determined using the Bradford assay using BSA of known concentration as a reference standard.

Identification of pIR23
Initial screening of potential reductive aminases was performed in the reverse direction utilizing the IREDy-go colorimetric screening assay as recently reported. [4] Ncinnamylcyclopropanamine was used as a representative substrate for oxidative deamination.
The top five IRED candidates were screened for activity in the reductive animation of cinnamaldehyde with allylamine. Reductive animation reactions were carried out in 100mM Tris pH 8.0 at a 500 ul scale in 2ml microcentrifuge tubes. The reaction mixture contained 10 mM cinnamaldehyde, 20 mM allyl amine, 4 mg mL -1 lyophilized IRED enzyme, 1 mg mL -1 lyophilized GDH enzyme (Codexis), 25mM glucose, and 0.5mM NADPH. Reactions where incubated at 30˚C for 24h, with shaking at 990rpm. Assays were quenched by the addition of 100 ul of 5M NaOH and vigorous mixed by vortexing. Samples were extracted into 1ml of MTBE following vigorous mixing and centrifugation1(3,000 x g for 1 minute), dried over anhydrous MgSO 4 and the now clear organic layer was subjected to GC-FID analysis.
GC analysis was performed on an Agilent 6850 GC system (Agilent, Santa Clara, CA, USA) with a flame ionization detector (FID) and autosampler. An HP-1 column with 0.32 mm inner diameter and 0.25 μm film thickness (Agilent, Santa Clara, CA, USA) was used, with the following analysis method: Inlet temperature 300˚C, 50:1 split, constant flow, Initial oven temp 100˚C, 2min hold, 30˚C per min ramp to isothermal point of 325˚C.

S5
Fig. S1. Comparison of product peaks obtained after GC analysis of biotransformation reactions performed with the identified five IRED hits, revealing IR23 as best candidate.

Identification of BacRedAm
Using sequences of fungal reductive aminases AspRedAm (XP_001827659.1) [5] and AdRedAm(EEQ92622.1) [5] , we performed blast a search against all non-reductant proteins sequences from Bacteria (taxid:2) available on the NCBI GenBank database as of September 2020. The search returned a sequence (PZN88780.1, now named BacRedAm) from a bacterium isolated from compost metagenome (Zoo Composter 4, Sao Paulo Zoo, Brazil) as the top hit (54% sequence identity, 95% query cover). The top 50 homologous bacterial sequences were further selected, and multiple sequence alignment performed using Muscle [6] showed that the relevant active site residues in fungal reductive aminases were conserved in BacRedAm. Hence, BacRedAm was selected for further studies. Initial activity screening and kinetic studies reveal similar activity profile with the fungal reductive aminases (Table 1), hence BacRedAm was selected as a prototype bacterial reductive aminase.

Determination of kinetic parameters
Specific activity (reductive aminase activity) was determined from initial rate measurements, monitoring NADPH depletion at 370 nm (ε = 2.216 mM−1 cm−1) using a Tecan infinite M200 microplate reader (Tecan Group, Switzerland). Steady-state kinetic measurements were performed with various concentrations of cinnamaldehyde (or hydrocinnamaldehyde) while the amine and NADPH concentrations were maintained at saturation. A typical reaction mixture contained aldehyde (0.05-20 mM), 80 mM amine nucleophile (added from buffer stock adjusted to pH 7.5), 0.5 mM NADPH, 1 % (v/v) DMSO and 5-10 µg of purified pIR23 or BacRedAM in a total volume of 200 µl (50 mM NaPi buffer, pH 7.5). The reaction was initiated by the addition of purified pIR23 or BacRedAm to the mixture. A unit of pIR23 or BacRedAm was equal to the amount of the pure enzyme required to consume 1 µmol NADPH per min.
Activity measurements were performed in triplicate and kinetic constants were determined through nonlinear regression based on Michaelis-Menten kinetics (QtiPlot software).

One-pot biotransformation for N-allylation of amines with acrylic acids
For the one-pot CAR-RedAm-mediated N-allylation of amines using acrylic acids to generate the corresponding allylic amines, a typical 500 uL reaction mixture contained 5 mM 1, 2% v/v DMSO, purified SrCAR (0.3mg mL -1 ), 0.4 mg mL -1 purified pIR23, 12.5 mM amine (20 mM for piperidine), 6.5 mM ATP, 10 mM MgCl 2 , 20 mM D-glucose, 0.6 mM NADP + , 0.3 mg mL -1 purified GDH, in 50 mM NAPi buffer, pH 7.5. Reaction mixtures in 2 mL Eppendorf tube were incubated at 30 °C with 250 rpm shaking for 18 h. The reaction was then basified with NaOH to pH >12; 500 uL of EtoAc added, vigorously mixed, centrifuged (15 °C, 13 000 rpm, 5 min); and organic layer collected. The remaining aqueous layer was then acidified to pH ~2 and further extracted with EtOAc with centrifugation (15 °C, 13 000 rpm, 10 min). The organic layers were combined and dried over anhydrous MgSO 4 and samples were analysed by GC-MS. S10 Figure S2. Plot and kinetic analysis from initial rate study of NADPH-dependent pIR23catalysed of reductive amination of cinnamaldehyde (left) or hydrocinnamaldehyde (right) with cyclopropylamine. Non-linear regression analysis was performed using QTiplot software. Figure S3. Plot and kinetic analysis from initial rate study of NADPH-dependent pIR23catalysed of reductive amination of cinnamaldehyde (left) or hydrocinnamaldehyde (right) with propylamine. Regression analysis was performed using QTiplot software. S11 Figure S4. Plot and kinetic analysis from initial rate study of NADPH-dependent pIR23catalysed of reductive amination of cinnamaldehyde (left) or hydrocinnamaldehyde (right) with proparglamine. Regression analysis was performed using QTiplot software. Figure S5. Plot and kinetic analysis from initial rate study of NADPH-dependent pIR23catalysed of reductive amination of cinnamaldehyde (left) or hydrocinnamaldehyde (right) with allylamine. Regression analysis was performed using QTiplot software. S12 Figure S6. Plot and kinetic analysis from initial rate study of NADPH-dependent pIR23catalysed of reductive amination of cinnamaldehyde (left) or hydrocinnamaldehyde (right) with methylammine. Regression analysis was performed using QTiplot software. Figure S7. Plot and kinetic analysis from initial rate study of NADPH-dependent pIR23catalysed of reductive amination of cinnamaldehyde (left) or hydrocinnamaldehyde (right) with benzylamine. Regression analysis was performed using QTiplot software. S13 Figure S8. Plot and kinetic analysis from initial rate study of NADPH-dependent pIR23catalysed of reductive amination of cinnamaldehyde (left) or hydrocinnamaldehyde (right) with piperidine. Regression analysis was performed using QTiplot software. Figure S9. Plot and kinetic analysis from initial rate study of NADPH-dependent pIR23catalysed of reductive amination of cinnamaldehyde (left) or hydrocinnamaldehyde (right) with pyrrolidine. Regression analysis was performed using QTiplot software.